Antigen recognition and thymic maturation of human TCR Vgamma9-Vdelta2 cells
Inauguraldissertation
zur
Erlangung der Würde eines Doktors der Philosophie vorgelegt der
Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel
von
Magdalena Kistowska aus Pozna ń , Polen
Basel 2007
Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät
auf Antrag von
Prof. Antonius Rolink (Fakultätsverantwortlicher)
Prof. Gennaro De Libero (Dissertationleiter)
Prof. Ed Palmer (Korreferent)
Basel, den 13.11.2007
Prof. Dr. Hans-Peter Hauri, Dekan
Niniejszą pracę dedykuję moim Rodzicom
(I dedicate this work to my Parents)
Acknowledgments
Foremost, I would like to thank my supervisor Gennaro for giving me the opportunity to work in his lab. Thanks for the guidance, support, inspiration and encouragement all throughout my thesis work. To have had a teacher with such a passion for science will surely play an important role in motivating me in my future career.
I also would like to thank everyone who shared with me the time in the institute, in particular those who worked with me in “Little Italy”. Special words of gratitude go to those who directly helped me with this work: Hans-Jürgen, Lucia, Lena and Nino.
Moreover I would like to thank my friends with whom I have shared unforgettable moments both within and outside of the lab:
Zaima, one of the most positive people I have ever met, for making beginning much easier;
Gabriel, the “coffee angel”, for always having something good to say;
Sabry, my hood-mate, for all the successful operations and for hours of valuable talking;
Manu, my “personal” biochemist, for all his help and for his contagious zest for life;
Fede, the “shopping queen”, for her almost unbreakable optimism that helped me in many moments;
Sami, the chemotaxis expert, for all the support;
In addition I would like to thank Vreni, the cell-sorter master and a good friend.
Last but not least I would like to thank one very special person, my best friend and life companion, Łukasz:
Dziękuję za to, Ŝe zawsze jesteś przy mnie...
Table of content
Abbreviations
10Summary
13Introduction
17Genetic organization of TCR γ and δ loci 17
Assembly of TCR γδ chains 19
TCR γδ structure 20
TCR γδ-CD3 complex 21
Development of TCR γδ cells 22
TCR γδ cell tissue distribution 27
TCR γδ stimulatory antigens 28
Natural non-peptidic phosphorylated antigens 28 Synthetic stimulatory ligands for TCR Vγ9-Vδ2 cells 32
Alkylamines 34
TCR Vγ9-Vδ2 antigen recognition 35
TCR γδ cells reactivity to MHC and MHC–like molecules 35
CD1c restricted TCR γδ cells 35
MIC and ULBP reactive TCR γδ cells 36
Other molecules 37
Stimulation by bacterial superantigens 38
Effector functions of TCR γδ cells 38
Role of TCR γδ cells in microbial infections 38
Tumor surveillance 41
Tissue homeostasis and repair 43
TCR γδ cells in autoimmune diseases and inflammation 44
Part 1
Intracellular endogenous ligands activating TCR V γ 9-V δ 2 cells
45Results 47
Active HMGR in tumor cells is required for activation of
TCR γδ cells 47
HMGR overexpressing cells are potent TCR γδ cells stimulators 50 Nitrogen-containing bisphosphonates treated APC activate
TCR γδ cells 51
nBP have different mechanisms of action than IPP 53 nBP require internalization for their activity 55 nBP induce accumulation of endogenous TCR γδ ligands 57 Identification of metabolites important for tumor cell recognition 59
Discussion 62
Part 2
Transient dysregulation of the mevalonate pathway during early bacterial infection leads to TCR V γ 9-V δ 2 cells activation
65Results 66
Stimulation of TCR Vγ9-Vδ2 cells by bacteria-infected APC is
MEP pathway independent 66
Endogenous mevalonate pathway is involved in generation
of TCR γδ ligands during infection 68
Bacterial infections modulate HMGR protein levels and
phosphorylation state 72
Increased PP2A activity leads to HMGR dephosphorylation
induced by bacterial infection 75
HMGR activity is increased during early times of
bacterial infection 77
Activity of MVK, PMVK and MVD is not changed during
bacterial infection 79
Increased HMGR activity during bacterial infection is MyD88
independent 80
Discussion 83
Part 3
Multi-drug related protein 5 (MRP5, ABCC5) is involved in
trafficking of phosphorylated mevalonate metabolites
88Results 88
Transfer of TCR γδ stimulatory ligands 88 Involvement of ATP-binding cassette transporter-C
(ABC-C) in transport of the TCR γδ ligands 90 MRP5 overexpression increases stimulation of TCR γδ cells 98 MRP5 downmodulation affects stimulation of TCR γδ cells 100
Discussion 104
Part 4
Thymic development of TCR V γ 9-V δ 2 cells
109Results 109
Localization of Tg T cell in lymphoid organs 109 Tg thymocytes have a semi-mature phenotype 112 Tg TCR is functional and induces T cell activation in vitro 114 DN TCR γδ thymocytes proliferate upon in vitro TCR stimulation 118 Triggering of Tg TCR in vivo induces maturation of Tg thymocytes 119 Upon TCR triggering TCR γδ T cells exit the thymus and colonize
peripheral lymphoid organs 122
Discussion 125
Conclusions
130Materials and methods
132Bacteria 132
Cell culture reagents 132
Cells 133
Freezing and thawing of primary cells and cell lines 133 Preparation of human monocytes and dendritic cells (DCs) 134 Expansion of human thymic epithelial cells (TEC) 134
Generation of human T cell clones 135
Maintenance of human T cell clones 135
T cell stimulation assays 136
Experiments with bisphosphonates 136
Bacterial infection experiments 136
Ligand transfer experiments 137
Experiments with drugs inhibiting transport proteins 137 Cytokine determination by Enzyme Linked Immunosorbent Assay
(ELISA) 139
Recombinant cytokines production 140
Generation of stable transfectants 140
Generation of MRP5 shRNA interference constructs 141
RT-PCR analysis of HMGR 142
Real-time quantitative PCR of MRP4 and MRP5 143
Immunoprecipitation of HMGR 144
Electrophoresis, transfer and western blotting 145
HMGR phosphorylation studies 147
PP2A activity assay 147
Calcium flux measurement 148
HMGR activity assay 148
LC-MS analysis of HMGR products 149
Mevalonate kinase, phosphomevalonate kinase and
diphosphomevalonate decarboxylase activity assays 150 Induction of mevalonate pathway products in cell lysates 151
Separation of the mevalonate metabolites by HPLC 152 Structural identification of the antigenic fraction by
mass-spectroscopy 153
14C-ZOL uptake 154
Mice 155
Screening of transgenic mice 156
Intrathymic injections 157
Preparation of mouse lymphoid cells 157
Preparation of mouse bone marrow derived dendritic cells 158
Activation assays with Tg T cells 158
Cell surface markers staining 158
Intracellular staining 159
Flow cytometry 159
Chemotaxis assay 160
Production of monoclonal antibodies from hybrydoma 161
Biotinylation of purified antibodies 162
Statistical analysis 162
References
163Appendix
189Functional CD1a is stabilized by exogenous lipids 189
Curriculum vitae
199Abbreviations
7-DHC 7-dehydrocholesterol
ABC ATP-binding cassette transporters
AP alkaline phosphatase
APC antigen presenting cell
APM antigen-presenting molecule
APS ammoniumpersulfate
ATP adenosine triphosphate
BrHPP bromohydrine pyrophosphate
BSA bovine serum albumin
CA calyculin A
CCR7 CC-chemokine receptor 7
CFTR cystic fibrosis transmembrane conductance regulator cDNA complemantary deoxyribonucleic acid
CDR complementarity determining region
cpm counts per minute
DC dendritic cells
DETC dendritic epithelial T cells
DIDS 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid DMAPP dimethylallylpyrophosphate
DMSO dimethylsulfoxide
DN double negative
DP double positive
EDTA ethylenediamine-tetraacetic acid ELISA Enzyme Linked Immunosorbent Assay FACS fluorescence activated cell sorting
FCS fetal calf serum
FPP farnesylpyrophosphate
GGPP geranylgeranylpyrophosphate
GM-CSF granulocyte-macrophage colony-stimulating-factor cGMP cyclic guanosine monophosphate
GPP geranylpyrophosphate
h hour(s)
HLA human leukocyte antigen
HMB-PP (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate HMGR 3-hydroxymethyl-3-glutaryl-CoenzymeA-reductase HPLC high performance liquid chromatography
HRP horse radish peroxidase
HS human serum
HSA human serum albumin
IELs intraepithelial lymphocytes
IFNγ interferon gamma
Ig immunoglobulin
IL interleukin
IPP isopentenylpyrophosphate
i.t. intra thymic
kDa kilo Dalton
KGF keratinocyte growth factor
LC-ESI-MS liquid chromatography-electrospray-mass spectrometry
mAb monoclonal antibody
MCT monocarboxylate transporters MEP 2-C-methyl-D-erythritol 4-phosphate
MEV mevastatin
MHC major histocompatibility complex
min minute(s)
MOI multiplicity of infection
Mon monensin
MDR multi-drug resistance protein MRP multi-drug related protein
MVD diphosphomevalonate decarboxylase
MVK mevalonate kinase
MVL mevalonolacton
nBP nitrogen-containing bisphosphonate drugs
NK natural killer
ND not determined
OA okadaic acid
OATP organic anion-transpoting polypeptide
PAM pamidronate
PBMC peripheral blood mononuclear cells PBS phosphate buffered saline solution PCR polymerase chain reaction
PHA phytohemagglutinin
PMSF phenylmethylsulfonylfluoride PMVK phosphomevalonate kinase Rag recombination activating gene
SBA sec-butylamine
SCID severe combined immunodeficiency
SDS sodiumdodecylsulfate
shRNA small hairpin RNA
SLC secondary lymphoid-tissue chemokine
SP single positive
SUR sulfonylurea receptors
TAP trasporter associated with antigen processing
TCR T cell receptor
TE Tris-EDTA buffer
TEC thymic epithelial cells
TEA triethylammonium-acetate
TEMED N,N,N',N'-tetramethylethylenediamine
Tg transgenic
TNFα tumor necrosis factor alpha
U unit(s)
Wt wild type
ZOL zoledronate
Summary
T cells are divided into two populations according to the type of TCR used for antigen recognition. One population uses a TCR heterodimer, which is
composed by the non-covalently associated alpha and beta chains. This TCR recognizes protein and lipid antigens, which are presented by MHC and CD1 antigen-presenting molecules, respectively. A second population uses a TCR heterodimer composed by the gamma and delta chains and recognizes non- peptidic ligands in the absence of MHC and CD1 restriction. In humans the major population of TCR γδ cells uses the Vγ9-Vδ2 TCR. This is a unique population because it is present only in primates and constitutes >50% of peripheral TCR γδ cells. TCR Vγ9-Vδ2 cells are activated by microbial phosphorylated metabolites and by so far unknown ligands expressed by a group of tumor cells. The principal microbial antigen is (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), an intermediate metabolite generated in 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoids biosynthesis.
Despite these cells were described in 1986, many aspects remain unclear, including the nature of the stimulatory ligands present in tumor cells, the mechanisms of their activation during infection, the molecular mechanisms involved in antigen presentation, and the requirements for thymic maturation. In this dissertation we have addressed these important issues using ex vivo cells, biochemical approaches for ligand identification, T cell activation assays and generation of transgenic mice expressing this human TCR.
We have identified endogenous metabolites generated in the mevalonate pathway as the tumor ligands which stimulate TCR Vγ9-Vδ2 lymphocytes. We have found that tumor cells show altered mevalonate pathway which leads to accumulation of intermediate metabolites. This is novel mechanism utilized by the immune system to monitor the metabolic integrity of cells and to react to those which have a dysregulation of this important metabolic pathway.
In a second series of studies we have investigated how TCR Vγ9-Vδ2 cells are activated during bacterial infections. Despite published studies identified HMB-PP as a potent stimulatory ligand in vitro, there was no formal evidence that this compound participates in cell activation during infection. Unexpectedly, we found that HMB-PP is not the major stimulatory ligand during infection and instead endogenous mevalonate metabolites are the stimulatory ligands. We describe how infection modifies the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), which is the key enzyme of the mevalonate pathway, and promotes increased synthesis of stimulatory metabolites. We show that infection induces a transient increase in HMGR protein levels and dephosphorylation, leading to increased enzymatic activity. This alteration occurs already within 1 hour after infection, thus representing a rapid mechanism reacting to infection. Thus, like with recognition of tumor cells, also during infection, the immune system of primates utilizes a mechanism which detects alterations of an important metabolic pathway.
We also investigated the mechanisms how mevalonate metabolites traffic within cells. We found that these ligands, which are generated within the
cytoplasm, are transported to the cell surface, where they interact with the TCR γδ, by the MRP5 transporter. We showed that MRP5-blocking drugs inhibit presentation to TCR γδ cells that over expression and knocking down of MRP5 protein increase and inhibit ligand presentation, respectively. These results show that like peptides, which are transported from cytoplasm to the ER through the ABC transporters TAP1 and TAP2, also TCR γδ ligands utilize ABC transporters to become immunogenic. We also found that MRP5 is not involved in forming complexes presented to the TCR γδ and that other unknown ubiquitous and non- polymorphic molecules are involved in this process.
In the last part of these studies we investigated the requirements for thymic maturation and peripheral expansion of TCR Vγ9-Vδ2 cells. We generated a transgenic (Tg) mouse model in which T cells express a TCR composed by human Vγ9-Vδ2 chains. Tg thymocytes express molecules characteristic of partially mature thymocytes together with high levels of Tg TCR. Tg cells do not acquire a mature phenotype and do not exit the thymus in the absence of TCR triggering. However, upon injection of TCR-specific mAbs, Tg thymocytes undergo maturation and colonize peripheral lymphoid organs. Mature Tg T cells remain in the periphery for up to 6 months, with a phenotype of naïve T cells and strongly react to physiological ligands when stimulated by human antigen-
presenting cells, which express the restriction element. Thus, Tg T cells expressing the human TCR Vγ9-Vδ2 resemble TCR αβ cells since they also require selection events during thymic maturation.
Our studies suggest that TCR Vγ9-Vδ2 cells by reacting to cells which accumulate mevalonate metabolites provide an early immune response during the time when antigen-specific TCR αβ cells have not yet been recruited and expanded. Thus, TCR γδ cells fulfill the role of sentinel cells which monitor the metabolic integrity of other cells. These cells undergo thymic selection events and require the presence of unique molecules for efficient antigen presentation.
Our studies indicate novel aspects of some of these important processes.
Introduction
TCR γδ cells represent a lymphocyte population phenotypically and functionally diverse from TCR αβ cells. They have been found in all vertebrates examined so far including humans (Brenner et al., 1986), monkeys (Malkovsky et al., 1992), mice (Saito et al., 1984; Raulet et al., 1991), rats (Lawetzky et al., 1990), rabbits (Isono et al., 1995), sheep (Hein and Mackay, 1991), cattle
(Mackay and Hein, 1989), horses (Schrenzel and Ferrick, 1995), pigs (Hirt et al., 1990; Saalmuller et al., 1990) and chicken (Bucy et al., 1988; Sowder et al., 1988).
They usually represent a small proportion (1-10%) of circulating
lymphocytes in most adult animals, while they represent a major proportion in certain extra-lymphoid sites. TCR γδ cells differ from classical MHC restricted T cells in terms of TCR diversity, requirements for antigen recognition and their role in immunity and tissue homoeostasis.
Genetic organization of TCR γγγγ and δδδδ loci
TCR γ and TCR δ chains genes, like TCR α, TCR β and immunoglobulins chain genes, are assembled during somatic rearrangement processes.
In human the TCR γ locus maps on chromosome 7 (Murre et al., 1985) and is composed of two constant gene segments (Cγ), five joining elements (Jγ) and fourteen variable (Vγ) genes of which six encode functional proteins and eight are pseudogenes (Lefranc and Rabbitts, 1990), (Figure 1A). These six genes can be
subdivided into two families: the VγI family composed of Vγ2, 3, 4, 5 and 8 genes and VγII consisting of Vγ9 (Vγ2 in other nomenclature) gene. The TCR γ chain undergoes Vγ-Jγ rearrangement and its variability at junctions is crated by addition of not germline encoded nucleotides (N nucleotides) during
recombination process by terminal deoxynucleotidyl transferase (Tdt), (Strauss et al., 1987; Huck et al., 1988).
Vα VαVα/δ Vα Vδ Dδ1-3 Jδ1-4 Cδ Vδ Jαn Cα
Vγ Jγ1 Cγ1 Jγ2 Cγ2
A
B
Vα VαVα/δ Vα Vδ Dδ1-3 Jδ1-4 Cδ Vδ Jαn Cα
Vγ Jγ1 Cγ1 Jγ2 Cγ2
A
B
Figure 1. Genetic organization of human (A) TCR γ locus and (B) TCR δ locus adapted from (Hayday, 2000). TCR V segments are green and pseudogens are grey; C
segments are red; J segments are blue and D segments are yellow. Only some Vγ and Vα are indicated.
TCR δ locus is closely linked to TCR α locus in contemporary mammals (Hayday et al., 1985; Hayday, 2000). In humans the δ locus is located within the TCR α locus on chromosome 14 (Collins et al., 1985), between Jα and Vα gene segments (Griesser et al., 1988), (Figure 1B). TCR δ chain is assembled via V-D- J rearrangement. The TCR δ locus is composed of single Cδ gene segment, four different Jδ segments preceded by three diversity (Dδ) elements (Chien et al.,
1987; Hata et al., 1987; Takihara et al., 1989). Some of V segments are used both as Vδ and Vα (Guglielmi et al., 1988; Takihara et al., 1989). Although eight distinct Vδ genes have been localized, only six of them were found expressed on the cell surface (Arden et al., 1995; Migone et al., 1995). The variability of TCR δ chain can be extremely high due to the Dδ segments that can undergo tandem rearrangement (Boehm et al., 1988) and flexible reading frame usage (Hata et al., 1988) creating diverse length and composition of the joining regions.
Therefore, despite the limited number of germline encoded elements for the TCR γδ, the potential repertoire of this TCR is at least three orders of magnitude higher than TCR αβ repertoire due to the extremely high variability in the CDR3 regions (Davis and Bjorkman, 1988; Hata et al., 1988). Comparison of the CDR3 length reveled that TCR γδ is more similar to immunoglobulins than to TCR αβ (Rock et al., 1994).
Assembly of TCR γγγγδδδδ chains
The particular feature of TCR γδ is the preferential association of Vδ chains with certain Vγ chains. In humans Vδ2 chain is usually associated with unique Vγ chain: Vγ9-JP-Cγ1 while Vδ1 and Vδ3 chains are mostly paired with various Vγ elements from VγI gene family using Cγ2 (Sturm et al., 1989; Hayday, 2000). Cγ1 gene segment usage allows formation of disulphate bond between TCR chains while usage of Cγ2 results in non-disulphate linkage (Krangel et al., 1987; Littman et al., 1987).
TCR γδ γδ γδ γδ structure
Crystal structure of human TCR Vγ9-Vδ2 revealed distinct differences as compared to TCR αβ or to antibody Fab fragments. TCR γδ has an unusual shape due to the small elbow angle and a small Vγ-Cγ inter-domain angle. The elbow angle, between pseudo two-fold symmetry axes that relate V to V and C to C (Lesk and Chothia, 1988), of TCR γδ is 110° while Fabs and TCR αβ have elbow angles in the range of 125-225° and 140-159°, respectively (Allison et al., 2001), (Figure 2).
Figure 2. TCR γδ and TCR αβ structures aligned to the V domains.
The C domains of TCR γδ are yellow while V domains are red. In the TCR αβ blue are the C domains and green are V domains. Black lines indicate pseudo two-fold symmetry axes. The values for elbow angles are indicated. PDB accession numbers for TCR γδ and TCR αβ are 1hxm and 1qsf, respectively.
The inter-domain angles, between the long axis of the C domains and long axis of the V domains, of TCR γδ have 42° for Cγ-Vγ and 101° for Cδ-Vδ. In contrast antibodies and TCR αβ have average inter-domain angles of 92° (VL-CL, σ=9°), 76° (VH-CH, σ=11°), 100° (Cα-Vα, σ=4°) and 67° (Cβ-Vβ, σ=3°).
Therefore, the 42° of Cγ-Vγ is the smallest inter-domain angle among these receptors. Moreover TCR γδ differ from TCR αβ also in the structure of C domains. The FG loop of Cγ is much smaller that the one of Cβ suggesting different binding with CD3ε subunit. The secondary structure of Cδ is composed of a regular immunoglobulin-like domain with three-stranded β-sheet as its outer face and therefore differs from the one or Cα (Allison et al., 2001)
The structural difference between TCR αβ and TCR γδ most likely reflects that these receptors recognize structurally different molecules.
TCR γγγγδδδδ-CD3 complex
The main difference between TCR αβ- and TCR γδ-CD3 complexes is their requirement for CD3δ chain. TCR γδ-CD3 complex, unlike TCR αβ, does not associate with CD3δ and contains only CD3γε dimers (Dave et al., 1997; Hayes and Love, 2002). The lack of CD3δ chains, unlikely for TCR αβ (Delgado et al., 2000), does not affect the ERK activation occurring upon TCR stimulation (Hayes and Love, 2002).
The difference in the recruitment of signaling molecules provides a difference in signaling potential of these T cell receptors. In fact TCR γδ cells,
upon CD3ε stimulation, have better proliferative response than TCR αβ cells (Hayes and Love, 2002). Therefore enhanced signaling capacity of TCR γδ cells together with their localization and recognition of native antigens allows these cells to respond rapidly and acquire effector functions faster than TCR αβ cells (Hiromatsu et al., 1992; Ferrick et al., 1995; Hayday, 2000; Hayes and Love, 2002).
Development of TCR γγγγδδδδ cells
TCR αβ and γδ cells develop in the thymus from the pluripotent CD34+ precursor cells deriving from bone marrow but distinct from stem cells (Res et al., 1996). In humans immature thymocytes can be divided accordingly to the
expression of CD34, CD38 and CD1a (Spits et al., 1998; Spits, 2002). The earliest thymic progenitors are CD34+CD38-CD1a-, followed by
CD34+CD38+CD1a- and CD34+CD38+CD1a+ cells, with CD1a expression correlating with T linage commitment (Sanchez et al., 1994). In the next stage cells start to express CD4, but not CD8, and they are referred to as CD4+ immature single positive (CD4 ISP) cells (Kraft et al., 1993; Spits, 2002).
Importantly, this population contains precursors for both TCR αβ and γδ cells meaning that these cells are before β-selection checkpoint (Ramiro et al., 1996;
Blom et al., 1999). The CD4 ISP stage is followed by cells that express CD4 and CD8α chain, and referred to as early double positive (EDP) cells (Spits, 2002).
Recently it has been shown that within the population of EDP there are still
present cells uncommitted to the linage. The TCR γδ developmental potential is only lost on double positive (DP) stage (Joachims et al., 2006).
Up to date there are no clear evidences that TCR γδ cells are undergoing selection process in the thymus. However, the TCR γδ repertoire generated in the human thymus is much more diverse than the one present in the periphery where TCR Vγ9 chain pairs only with TCR Vδ2 chain (Casorati et al., 1989; Krangel et al., 1990), thus suggesting that certain selection of TCR γδ cells takes place in the thymus.
More extensive studies concerning TCR γδ cells development have been performed using mouse models. In mice TCR γδ cells and TCR αβ cells also develop from a common thymic double negative (DN) precursor but they diverge into separate lineages very early in ontogeny (Petrie et al., 1992; Dudley et al., 1995).
Immature αβ lineage cells expressing pre-TCR undergo proliferation and transition to CD4 CD8 DP stage (Fehling et al., 1995). DP thymocytes which express mature TCR αβ, follow positive and/or negative selection and emerge as CD4 or CD8 single positive (SP) thymocytes (Fehling et al., 1995 ). In contrast γδ lineage cells during differentiation do express mature TCR γδ complex, remain mainly DN and undergo limited proliferation (Pardoll et al., 1988).
The developmental stage at which γδ lineage diverges from αβ lineage presumably occurs between the CD44+CD25+ (DN2) and CD44-CD25- (DN4) stage (Shortman et al., 1991; Petrie et al., 1992; Kang et al., 2001). There are indications that TCR γδ-dependent developmental checkpoint take place already
at DN3 stage (Prinz et al., 2006; Taghon et al., 2006), (Figure 3). Progression through this checkpoint is marked by high expression of CD27 on TCR γδ cells (Taghon et al., 2006).
DN1
DN1 DN2DN2 DN3aDN3a
DN3b DN3b
DN3b DN3b
γδγδ γδγδ γδγδ γδγδ
DN4
DN4 DPDP
CD4+ CD4+
CD8+ CD8+
TCRγ, TCRδ, TCRβ rearrangements
TCRαrearrangement γδ-selection
β-se
lection TCR αβ-positive
selection
Figure 3. Schematics of thymic T cell development of TCR αβ and TCR γδ cells modified from (Hayday and Pennington, 2007).
TCR γ and δ gene rearrangements are initiated at DN2 stage whereas
rearrangement of TCR β gene is slightly delayed and begins between DN2 and DN3 stages (Livak et al., 1999).
The αβ/γδ lineage choice is mediated by single TCR and regulated by the strength of TCR signal (Hayes et al., 2003; Haks et al., 2005; Hayes et al., 2005) and by Notch signaling (Garbe and von Boehmer, 2007). Strong TCR signals through TCR γδ or TCR αβ direct either the development of DN γδ lineage cells or MHC independent development of TCR αβ cells with γδ DN phenotype
(Terrence et al., 2000; Garbe and von Boehmer, 2007), (Figure 4A). Instead weak TCR signals (from pre-TCR or TCR γδ or TCR αβ) in synergy with Notch signaling would favor the αβ lineage commitment (Figure 4B), (Garbe and von Boehmer, 2007).
TCR γδγδγδγδ TCR γδγδγδγδ
TCR αβαβαβαβ TCR αβαβαβαβ
DN
strong TCR signaling weak TCR signaling and Notch signaling
pre-TCR TCR αβαβαβαβ
DP
TCR γδγδγδγδ
TCR αβαβαβαβ
A B
DN
Figure 4. Influence of TCR signal strength and Notch signaling on the αβ/γδ lineage choice adapted from (Garbe and von Boehmer, 2007).
(A) Strong signaling by the TCR γδ or the TCR αβ results in differentiation into DN γδ lineage cells or DN cells with a γδ lineage phenotype but TCR αβ expression.
(B) Weak signal by the TCR γδ, pre-TCR or TCR αβ together with Notch signaling results in differentiation into αβ lineage cells.
Recently involvement of Sox13 transcription factor in regulation of αβ/γδ lineage differentiation has been shown. Sox13 is essential for the proper development of TCR γδ cells, but not TCR αβ cells. It is highly expressed on DN1 and DN2 cells and subsequently downregulated in αβ-lineage differentiating cells. Significant
levels of Sox13 are maintained in peripheral TCR γδ cells. Since Sox 13 expression by DN2 cells is heterogenous (50% of DN cells are positive for Sox13) it has been suggested that some lineage separation occurs even before TCR rearrangement (Melichar et al., 2007).
TCR γδ cells developing in adult, but not fetal thymus, require a significant number of DP thymocytes which trans regulate differentiation of TCR γδ cells through involvement of transcription factor RORγt and lymphotoxin β receptor (LTβR). The proper signaling from LTβR is essential for correct TCR γδ biased gene expression which is required for the proper function of TCR γδ cells. Thus the trans conditioning overall influences rather cell’s functional competences than commitment to the lineage (Pennington et al., 2003; Silva-Santos et al., 2005;
Hayday and Pennington, 2007).
Another factor important for the proper development of TCR γδ cells is IL-7 receptor signaling which promotes the expansion and survival of TCR γδ cells in the thymus. Moreover, it is also required for the proper recombination of TCR γ locus (Ikuta et al., 2001).
An important question which remains to be answered is whether TCR γδ requires ligand engagement for the proper TCR γδ cells development. Up to date only indirect evidence suggest the requirement for ligand-mediated positive selection in fetal thymus of the dendritic epidermal T cells (DETC), (Xiong et al., 2004; Lewis et al., 2006). This subset of TCR γδ cells populates mouse skin.
TCR γγγγδδδδ cell tissue distribution
TCR γδ cells comprise 1-5% of circulating T cells while they are very abundant in tissues. In human TCR γδ cells expressing TCR Vδ1 or Vδ3 chains are predominant in the epithelium of the intestine (De Libero et al., 1993) where they comprise the majority of intraepithelial lymphocytes (IELs). Cells bearing TCR Vδ2 chain localize mainly in secondary lymphoid organs, tonsils and
peripheral blood where they represent 5-10% of total lymphocytes (Casorati and Migone, 1990; Parker et al., 1990; Haas et al., 1993). Importantly, in humans and some primates about 50-80% of all TCR γδ express restricted TCR composed by the Vγ9 and Vδ2 chains (Porcelli et al., 1991). In the postnatal thymus TCR Vγ9- Vδ2 cells constitute a minor population (up to 1% of total thymocytes), (Casorati et al., 1989; Falini et al., 1989) but they expand in the periphery, probably due to the continuous stimulation by unknown factors (Parker et al., 1990).
Also in mice certain populations of TCR γδ cells localize in epithelia of particular organs. In the epidermis DETC account for ~100% of resident IELs.
They have TCR composed of Vγ3 and Vδ1 chains lacking junctional diversity (Havran and Allison, 1990). TCR Vγ4 chain is predominantly expressed in the reproductive tract, lung and tongue (Itohara et al., 1990). In the small intestine TCR γδ cells preferentially use Vγ5 and Vγ1.1 chains (Pereira et al., 2000) while in the secondary lymphoid organs TCR γδ cells mainly express Vγ2, Vγ1.1 and Vγ1.2 chains (Raulet et al., 1991; Pereira et al., 2000).
The tissue specific distribution of cells expressing particular TCR γδ heterodimers most likely is associated with recognition of ligands present at the localization site.
TCR γγγγδδδδ stimulatory antigens
Natural non-peptidic phosphorylated antigens
The main population of human TCR γδ cells expressing TCR Vγ9-Vδ2 respond in vitro to pathogen derived (both bacterial and parasite) extracts (Morita et al., 2000). The stimulatory components, obtained form mycobacteria cell lysates, have small molecular weight (less than 500 Da), are protease-resistant and contain critical phosphate residue (Pfeffer et al., 1990; Constant et al., 1994;
Schoel et al., 1994; Tanaka et al., 1994). The analysis of Mycobacterium smegmatis culture supernatants resulted in identification of
isopentenylpyrophosphate (IPP) and its hydroxymethyl derivatives as natural TCR γδ ligands (Tanaka et al., 1995). These compounds are intermediate metabolites of isoprenoids biosynthesis. In eukaryotes, archaebacteria and certain eubacteria the biosynthesis of IPP proceeds via mevalonate pathway, while in many eubacteria and plastids of algae and higher plants, IPP is supplied by 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (Eberl et al., 2003), (Figure 5 and Table 1).
Mevalonate pathway
acetyl CoA + acetoacetyl CoA
HMG CoA
mevalonate
mevalonate phosphate
mevalonate pyrophosphate
isopentenylpyrophosphate (IPP)
geranylpyrophosphate (GPP)
farnesylpyrophosphate (FPP)
cholesterol
geranylated proteins farnesylated
proteins other products
HMG CoA reductase
dimethylallylpyrophosphate (DMAPP)
FPP-synthase
geranylgeranylpyrophosphate (GGPP)
B
mevalonate kinase
phosphomevalonate kinase
diphosphomevalonate decarboxylase
MEP pathway
pyruvate + glyceraldehyde-3-phosphate
1-deoxy-D-xylulose-5-phosphate (DOXP)
2-C-methyl-D-erythritol-4-phosphate (MEP)
4-hydroxy-3methyl-but-2-enyl-pyrophosphate (HMB-PP)
dimethylallylpyrophosphate (DMAPP) isopentenylpyrophosphate
(IPP)
Dxs
Dxr
YgbP YchB YgbB GcpE
LytB
Ipi
A
Mevalonate pathwayacetyl CoA + acetoacetyl CoA
HMG CoA
mevalonate
mevalonate phosphate
mevalonate pyrophosphate
isopentenylpyrophosphate (IPP)
geranylpyrophosphate (GPP)
farnesylpyrophosphate (FPP)
cholesterol
geranylated proteins farnesylated
proteins other products
HMG CoA reductase
dimethylallylpyrophosphate (DMAPP)
FPP-synthase
geranylgeranylpyrophosphate (GGPP)
B
mevalonate kinase
phosphomevalonate kinase
diphosphomevalonate decarboxylase
MEP pathway
pyruvate + glyceraldehyde-3-phosphate
1-deoxy-D-xylulose-5-phosphate (DOXP)
2-C-methyl-D-erythritol-4-phosphate (MEP)
4-hydroxy-3methyl-but-2-enyl-pyrophosphate (HMB-PP)
dimethylallylpyrophosphate (DMAPP) isopentenylpyrophosphate
(IPP)
Dxs
Dxr
YgbP YchB YgbB GcpE
LytB
Ipi
A
Figure 5. Schematics of prokaryotic MEP and eukaryotic mevalonate pathways of isoprenoids biosynthesis.
(A) MEP pathway with indicated genes coding enzymes.
(B) Mevalonate pathway with indicated selected enzymes.
Organism MEP pathway Mevalonate pathway Prokaryotes
Eubacteria
Mycobacterium tuberculosis + -
Mycobacterium leprae + -
Escherichia coli + -
Haemophilus influenzae + -
Chlamydia pneumoniae + -
Pseudomonas aeruginosa + +
Listeria monocytogenes + +
Staphylococcus aureus - +
Streptococcus pneumoniae - +
Borrelia burgdorferi - +
Archaebacteria
Pyrococcus horikowskii - +
Methanobacterium - +
Eukaryotes
Apicomplexan parasites
Plasmodium farciparum + -
Plants
Arabidopsis thaliana + +
Fungi
Saccharomyces ceravisiae - +
Mammals - +
Table 1. Utilization of MEP and mevalonate pathways by selected organisms, adapted from (Morita et al., 2000).
Beside IPP also other mevalonate pathway metabolites, including dimethylallylpyrophosphate (DMAPP), farnesylpyrophosphate (FPP),
geranylgeranylpyrophosphate (GGPP) and geranylpyrophosphate (GPP), (Burk et al., 1995; Tanaka et al., 1995; Morita et al., 1999), (Figure 6) behave as TCR Vγ9-Vδ2 cells ligands, although with a potency 30-300 times lower than that IPP.
Furthermore, an antigenic intermediate of bacterial MEP pathway, (E)-4-hydroxy- 3-methyl-but-2-enyl pyrophosphate (HMB-PP) was identified as very strong TCR
γδ antigen (Hintz et al., 2001), (Figure 6). The reactivity of human peripheral blood mononuclear cells (PBMC) towards HMB-PP is restricted to TCR Vγ9-Vδ2 cells and leads to upregulation of activation markers, secretion of pro-
inflammatory cytokines and expansion of these TCR γδ cells (Hintz et al., 2001;
Eberl et al., 2002; Reichenberg et al., 2003).
The stimulatory capacity of HMB-PP for TCR Vγ9-Vδ2 cells is approximately four orders of magnitude higher than IPP. Therefore it was suggested that HMB-PP is the key activator of TCR Vγ9-Vδ2 cells during
bacterial infection (Eberl et al., 2003). This aspect will be further discussed in this work.
Figure 6. Structures of selected natural prenyl pyrophosphates.
Synthetic stimulatory ligands for TCR Vγ9-Vδ2 cells
Up to date a number of synthetic compounds (Figure 7) that are able to stimulate TCR Vγ9-Vδ2 cells have been synthesized, including methyl
phosphate, monoethyl pyrophosphate (Tanaka et al., 1994), bromhydrin pyrophosphate (BrHPP, PhosphstimTM), (Belmant et al., 2000; Espinosa et al., 2001) and 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP), (Tanaka et al., 2007). These compounds specifically activate TCR Vγ9-Vδ2 cells which upon stimulation release cytokines and induce target cell lysis (Belmant et al., 2000).
As compared with naturally occurring ligands synthetic compounds are very strong agonist i.e. BrHPP have half-maximal activity in the nano molar concentration range (Belmant et al., 2000; Espinosa et al., 2001). Currently BrHPP and 2M3B1PP are produced in large scale amounts and are being used for clinical trials.
Figure 7. Structures of selected stimulatory synthetic phosphorylated antigens.
Another group of synthetic compounds are nitrogen-containing
bisphosphonates (nBP). There drugs prevent bone resorption and are used for treatment of Paget’s disease (Hosking, 2006), tumor-associated bone diseases (Sauty et al., 1996; Berenson et al., 2001; Tripathy et al., 2004) and osteoporosis (Boonen et al., 2005). They induce selective expansion of TCR Vγ9-Vδ2 cells in peripheral blood of human beings after intravenous administration (Kunzmann et al., 2000). Activated by nBP TCR γδ cells secrete pro-inflammatory cytokines and kill target tumor cells in vitro (Kunzmann et al., 2000). Structural similarities of nBP and bacterial antigens suggested that they might mimic natural ligands (Kunzmann et al., 1999), (Figure 8).
Figure 8. Structures of selected biological active nBP.
Importantly, nBP by blocking farneslypyrophosphate-synthase (FPP- synthase), (Thompson et al., 2002) one of the enzymes in the mevalonate pathway (Figure 5), lead to accumulation of IPP/DMAPP in vitro (Bergstrom et
al., 2000). Therefore, nBP could increase levels of intracellular ligands responsible for activation of TCR γδ cells (see this work).
Alkylamines
Different class of compounds activating TCR Vγ9-Vδ2 cells in an antigen specific manner are alkylamines (Bukowski et al., 1999). These small organic molecules contain short (two to five carbon atoms) alkyl chain linked to positively charged amine group (Figure 9). Alkylamines are produced and secreted by several bacterial strains (Daneshvar et al., 1989; Bukowski et al., 1999). They were also found in plants (apples), (Hartmann, 1967) and plant products (tea or wine), (Asatoor, 1966; Ibe et al., 1991). In order to stimulate TCR γδ cells, alkylamines must be present in milli molar concentrations which are unlikely present in vivo. This raises the question of the physiological relevance of alkylamines of TCR γδ activation and of the mechanisms how they are active.
Figure 9. Structures of selected alkylamines.
TCR Vγγγγ9-Vδδδδ2 antigen recognition
TCR Vγ9-Vδ2 cells are activated in a crossreactive manner by variety of ligands like IPP, DMAPP, 2,3-diphosphogyceric acid (DPG), glycerol-3-
phosphate (G3P), xylose-1-phosphate (Xyl-1P) , ribose-1-phosphate (Rib-1-P), (Burk et al., 1995). Optimal stimulation of TCR γδ cell is driven by cells of human origin (De Libero et al., 1991). The recognition of phosphate-containing antigens requires cell-cell interactions without the need for antigen processing (De Libero et al., 1991; Kabelitz et al., 1991; Lang et al., 1995; Morita et al., 1995). Taken together this suggests the existence of a dedicated antigen-presenting molecule.
TCR Vγ9-Vδ2 cells recognize phosphorylated antigens in the absence of MHC or CD1 restriction since APC lacking MHC class I, β2-microglobulin, CD1, or MHC class II molecules are able to activate TCR Vγ9-Vδ2 cells (Morita et al., 1995).
Moreover, tumor cell lines and normal PBMCs are able to activate TCR Vγ9-Vδ2 cells form different donors (Morita et al., 1995). Thus, the putative antigen- presenting molecule has rather limited or no polymorphism and is constitutively expressed in a variety of tissues.
TCR γγγγδδδδ cells reactivity to MHC and MHC–like molecules CD1c restricted TCR γδ cells
Human TCR γδ composed of Vδ1 chain paired with either Vγ1 or Vγ2 chains recognize CD1c molecule which is mainly expressed on immature DC and B cells (Faure et al., 1990; Spada et al., 2000). CD1c is a member of CD1 family molecules which are non-MHC-encoded proteins sharing structural similarities
with MHC class I molecules. CD1 antigen-presenting molecules have little polymorphism and are specialized in the presentation of lipids and glycolipids (Beckman et al., 1994; Matsuda and Kronenberg, 2001; De Libero and Mori, 2003).
This recognition occurs in the absence of exogenous foreign antigen (Spada et al., 2000; Vincent et al., 2002) suggesting reactivity against self-lipid loaded into CD1c molecule (De Libero and Mori, 2003). TCR γδ cells upon recognition of CD1c secrete pro-inflammatory cytokines which contribute to the DC maturation process (Ismaili et al., 2002; Leslie et al., 2002). In addition, they have a cytolytic, Th1 effector phenotype and produce granulysin (Spada et al., 2000).
MIC and ULBP reactive TCR γδ cells
Subset of TCR Vδ1+ cells have been found to interact with cells expressing stress-induced MHC class I-related chain (MICA and MICB)
molecules (Groh et al., 1998) and UL16-binding protein (ULBP family) molecules (Poggi et al., 2004). MIC are encoded within the MHC locus (Bahram et al., 1994) while ULBPs are encoded by genes on chromosome 6q25. MIC proteins are upregulated in response to cellular stress like heat shock in intestinal epithelium and on epithelial tumors (Groh et al., 1996; Groh et al., 1999) while ULBPs are expressed on various cancers of hematopoietic origin including acute myeloid and lymphoblastic leukemias (Salih et al., 2003).
Despite the structural similarities between MIC and MHC class I molecules, MIC do not associate with β2-microglobulin and do not present peptides, probably due to the limited size of the putative peptide binding grove and conformational differences in α1 and α2 domains (Groh et al., 1998; Li et al., 1999). TCR Vδ1+ cells interact with MIC trough the TCR (TCR-dependent signal 1) and through natural killer activating receptor NKG2D (NKG2D dependent costimulatory signal 2), (Wu et al., 2002) while ULBPs most likely interact only through NK receptors (Cosman et al., 2001).
Other molecules
Among human TCR γδ cells a few T cell clones, expressing TCR Vδ1 chain, react to allo–MHC molecules including HLA-A2 (Spits et al., 1990), HLA- A24 (Ciccone et al., 1989), HLA-B27 (Del Porto et al., 1994) and some
unspecified class I molecules (Rivas et al., 1989). In al these studies a cognate interaction of the TCR γδ with MHC molecules was not formally demonstrated.
In mouse TCR γδ cells recognizing T10/T22 MHC class Ib molecules (Matis et al., 1989; Van Kaer et al., 1991) or I-Ek MHC class II molecule (Matis et al., 1989) have been described. Recognition of these molecules is independent from the peptide binding and antigen processing (Schild et al., 1994; Crowley et al., 2000). In the case of I-Ek recognition depends on the post-translational changes in its glycosilation (Hampl et al., 1999).
Stimulation by bacterial superantigens
Bacterial superantigens are toxins secreted by several bacterial species, which activate T cells by binding to the non-polymorphic region of MHC class II molecules outside of the antigen binding groove (Kozono et al., 1995) and to the Vβ domain of the TCR αβ (Li et al., 1998). TCR Vγ9+ cells are activated by the superantigen staphylococcal enterotoxin A (SEA), (Loh et al., 1994; Morita et al., 2001). This stimulation occurs in a MHC class II dependent but Vδ independent manner. TCR γδ cell require higher concentrations of SEA for stimulation as compared to TCR αβ cells (Morita et al., 2001). Therefore, due to the structural similarity between TCR Vγ and TCR Vβ (Allison et al., 2001) and comparable concentrations required for T cell stimulation (Surman et al., 1994) interaction of Vγ with SEA might resemble that of Vβ with staphylococcal enterotoxin B.
Effector functions of TCR γγγγδδδδ cells
Role of TCR γδ cells in microbial infections
TCR γδ cells were found to expand to high levels during a variety of bacterial, viral and protozoan infections. Elevated levels of TCR Vγ9-Vδ2 cells in the peripheral blood were observed in patients infected with Mycobacterium tuberculosis (Barnes et al., 1992), Mycobacterium leprae (Modlin et al., 1989), Listeria monocytogenes (Jouen-Beades et al., 1997), Francisella tularensis (Poquet et al., 1998), Brucella melitensis (Bertotto et al., 1993), Salmonella typhimurium (Hara et al., 1992), Ehrlichia (Caldwell et al., 1995). Also patients with the following parasite infections have increased number of TCR γδ cells:
Leishmania donovani (Raziuddin et al., 1992), Toxoplasma ssp (Scalise et al., 1992), Plasmodium falciparum (Ho et al., 1990). Moreover TCR Vγ9-Vδ2 cells may increase in case of Epstein-Barr virus (EBV), (De Paoli et al., 1990) and Herpes simplex virus (HSV), (Bukowski et al., 1994) infections.
During certain microbial infections TCR Vγ9-Vδ2 cells expand even up to 50-fold (Table 2).
Infectious disease % of TCR γδ cells Reference bacterial
tuberculosis 14 (35) (Balbi et al., 1993)
tularemia 33 (Sumida et al., 1992; Poquet et al., 1998) salmonellosis 18 (48) (Hara et al., 1992)
brucellosis 29 (48) (Bertotto et al., 1993) ehrlichiosis 57 (97) (Caldwell et al., 1995) H. influence/meningitis 27 (37) (Raziuddin et al., 1994) N. meningititis/meningitis 25 (42) (Raziuddin et al., 1994) S. pneumoniae/meningitis 35 (46) (Raziuddin et al., 1994)
legionellosis 15 (Kroca et al., 2001) listeriosis 12 (33) (Jouen-Beades et al., 1997) Coxiella brunetii/Q-fever 16 (30) (Schneider et al., 1997) parasite
acute malaria (non endemic) 18 (46) (Schwartz et al., 1996) toxoplasmosis 9 (15) (Scalise et al., 1992) leishmaniases 13 (18) (Russo et al., 1993)
Table 2. Examples of human TCR γδ cells expansion in response to infection.
Percentage of TCR γδ cells shows mean values detected in patients. Maximal number of detected cells is shown in brackets.
Expanded and activated TCR Vγ9-Vδ2 cells may directly participate in anti-microbial immune responses inducing killing of bacteria, through granulysine release, and bacteria-infected cells, through preforin and/or Fas-Fas ligand interactions (Hara et al., 1992; Dieli et al., 2000; Ottones et al., 2000). Activated TCR Vγ9-Vδ2 cells are able to produce significant amounts of Th1 cytokines:
IFNγ and TNFα providing an important stimulus for macrophages attraction during the early stage of infection (Garcia et al., 1997; Wang et al., 2001).
Moreover, TCR Vγ9-Vδ2 cells release large quantities of the β-chemokines such as macrophage inflammatory protein-1α (MIP-1α, CCL3) and MIP-1β (CCL4), (Cipriani et al., 2000). In vitro MIP-1α and MIP-1β attract TCR αβ CD4+ and TCR αβ CD8+ cells, respectively (Schall et al., 1993; Taub et al., 1993). Therefore, chemokines release by TCR Vγ9-Vδ2 cells might contribute to the pro-
inflammatory microenvironment at the sites of infection.
The levels of TCR Vδ1/Vδ3 are elevated in renal allograph recipients developing cytomegalovirus (CMV) infection (Dechanet et al., 1999a). Moreover TCR Vδ2- cells activated by CMV-infected fibroblasts produce large amounts of TNFα and kill infected cells. This recognition is mediated by TCR independently of MHC class I presentation and without NKG2D engagement (Halary et al., 2005). Infection with human immunodeficiency virus-1 (HIV-1) leads to
proliferation of TCR Vδ1 cells in the peripheral blood (Hinz et al., 1994). Most likely, these TCR γδ cells are activated in the intestinal epithelia and then migrate to the peripheral blood (Dechanet et al., 1999b).
In mice TCR γδ cells expand in response to mycobacteria (Janis et al., 1989), listeria (Hiromatsu et al., 1992) and salmonella (Emoto et al., 1992)
infections. Importantly, mice lacking TCR γδ cells develop enhanced inflammation characterized by disruption of macrophage homeostasis and liver necrosis
(Carding and Egan, 2002) and they do not survive infections with Listeria monocytogenes (Skeen et al., 2001) or Klebsiella pneumoniae (Moore et al., 2000). Furthermore, in TCR αβ deficient mice, TCR γδ cells provide early protective immune responses against listeriosis (Mombaerts et al., 1993) and malaria (Tsuji et al., 1994).
Tumor surveillance
TCR Vγ9-Vδ2 cells have been shown to recognize and kill tumor
transformed cells like B cell lymphomas (Fisch et al., 1990; Selin et al., 1992), thymic lymphomas (De Libero et al., 1991) and erythroleukemia cells (Di Fabrizio et al., 1991). Moreover, this population of TCR γδ cells is expanded in blood and/or intra-lesions of patients with hemopoietic and solid tumors (Bonneville and Fournie, 2005).
The potent anti-tumor activities of TCR γδ cells have recently stimulated great interest in of TCR γδ cells cell-based cancer immunotherapy. TCR Vγ9-Vδ2 cells expanded ex vivo, with BrHPP and IL-2 and derived from patients with metastatic form of renal cell carcinoma have the ability to kill autologus primary renal tumor cells (Viey et al., 2005). Furthermore, nBP activated TCR Vγ9-Vδ2 cells produce cytokines, exhibit specific cytotoxicity against myeloma cell lines,
and lead to reduced survival of autologous myeloma cells (Kunzmann et al., 2000). TCR Vγ9-Vδ2 cells expanded in vitro in the presence of aledronate (another nBP) and IL-2 maintain their anti-tumor activity in vivo after adoptive transfer into mice with severe combined immunodeficiency (SCID), (Kabelitz et al., 2004).
Promising results in the treatment of patients with low-grade non-Hodgkin lymphoma and multiple myeloma were achieved by in vivo stimulation of TCR γδ cells using pamidronate and low-dose IL-2. This type of immunotherapy resulted in tumor regression (Wilhelm et al., 2003).
TCR Vγ9-Vδ2 cells have a phenotype of memory cells (Miyawaki et al., 1990; De Rosa et al., 2004) and the capacity to promptly release IFNγ and TNFα.
These characteristics implicate that they can be rapidly recruited to the site of tumorgenesis and therefore contribute to early immune protection.
Human TCR Vδ1 cells exhibit a selective lytic activity against various tumor cell lines like colorectal cancer, esophageal cancer, renal cell cancer, pancreatic cancer, lung cancer (Ferrarini et al., 1994; Zocchi et al., 1994;
Choudhary et al., 1995; Maeurer et al., 1996; Groh et al., 1999; Thomas et al., 2001). The recognition of tumor cells occurs via MIC or ULBP molecules which interact directly with NKG2D and possibly with TCR present on TCR Vδ1 cells (Groh et al., 1999; Wu et al., 2002; Poggi et al., 2004). Recently, it was reported that upon MICA-NKG2D interactions the antigen-dependent effector functions of TCR Vγ9-Vδ2 cells can be enhanced (Das et al., 2001a).
Tissue homeostasis and repair
TCR γδ cells from both human and mouse are able to produce
keratinocyte growth factor (KGF), (Boismenu and Havran, 1994; Workalemahu et al., 2003), a cytokine promoting epithelial cell growth (Visco et al., 2004). Human TCR γδ cells from bronchoalveolar lavage in the presence of IPP are able to secrete fibroblast growth factor 9 (FGF-9), associated with epithelial cell proliferation (Workalemahu et al., 2004).
Furthermore, upon activation with IPP TCR γδ cells secrete metalloproteinase 7 (MMP7) which serves a key role in epithelium repair (Workalemahu et al., 2006).
Therefore TCR γδ cells contribute to the maintenance of epithelial homeostasis and play a role in the restoring epithelial integrity.
Mouse DETCs can be activated in vitro by stressed or damaged
keratinocytes. Upon activation they secrete KGF which implicates their role in the response to skin damage. They also participate in keratinocyte survival by
constitutively secreting insulin-like growth factor 1 (IGF-1). In addition TCR δ-/- mice have problems with tissue repair and keratinocyte homeostasis. The lung and intestine TCR γδ cells, exhibit similar roles in the maintenance epithelium integrity (Witherden et al., 2000; Jameson and Havran, 2007).
The fact that TCR γδ cells reside in epithelial tissues of all mammals, suggest that they play a conserved role in the monitoring of tissue integrity.
TCR γδ cells in autoimmune diseases and inflammations
There are findings suggesting that TCR γδ cells could be involved in the pathogenesis of autoimmune diseases. Accumulation of TCR γδ cells has been observed in the inflamed synovium of patients with rheumatoid arthritis
(Holoshitz, 1999). By using a mouse model of collagen induced arthritis it was demonstrated that TCR γδ cells can have pro-inflammatory functions early in the disease but anti-inflammatory during the late stage of the disease (Peterman et al., 1993).
The number of TCR Vγ9-Vδ2 cells is increased in the peripheral blood of patients with diabetes mellitus (Lang et al., 1991) and in pre-diabetic and diabetic children (Gyarmati et al., 1999). The regulatory role of TCR γδ cells in diabetes was
studied by using non-obese diabetic (NOD) mouse model. The intranasal
inhalation of pro-insulin leads to the generation of a population of regulatory TCR γδ cells that can suppress the development of diabetes (Harrison et al., 1996).
In case of patients with multiple sclerosis (MS) elevated levels of TCR Vδ1 cells were found in the brain lesions (Wucherpfennig et al., 1992) and cerebrospinal fluid (Nick et al., 1995). The same population of TCR γδ cells has been found to be increased in the blood and intestine of patients with inflammatory bowel disease (Soderstrom et al., 1996; McVay et al., 1997).
Part 1
Intracellular endogenous ligands activating TCR V γγγγ 9-V δδδδ 2 cells
(These results have been published in The Journal of Experimental Medicine, 2003, 197, 163-168)
TCR Vγ9-Vδ2 cells recognize bacteria phosphorylated metabolites and also react to bone marrow-derived tumor cells such as Daudi Burkitt’s lymphoma line (Fisch et al., 1990; Malkovska et al., 1992). In order to identify the nature of the tumor antigens we investigated whether TCR γδ cells recognize
phosphorylated nonpeptidic ligands resembling those produced by microbes.
One of the potent identified bacterial antigens is IPP, an intermediate product of isoprenoids biosynthesis which is present in prokaryotic as well as in eukaryotic cells (Burk et al., 1995; Tanaka et al., 1995). Bacteria produce IPP in either MEP or mevalonate pathways (Morita et al., 2000), (Figure 5), whereas in eukaryotes IPP is generated exclusively in the mevalonate pathway (Figure 5). It was previously reported that in some hematological malignancies (Harwood et al., 1991) and mammary carcinomas (Asslan et al., 1999) expression and function of 3-hydroxymethyl-3-glutaryl-CoenzymeA-reductase (HMGR), the rate-limiting enzyme in the mevalonate pathway, is increased. Based on these findings we investigated whether dysregulation of mevalonate pathway in tumors may lead to accumulation of mevalonate metabolites thus resulting in activation of TCR Vγ9-
Vδ2 cells. In the following studies we took advantage of drugs that influence enzymes of the mevalonate pathway (Figure 10).
Figure 10. Mevalonate pathway with indicated compounds used in this study and the affected enzymes.
Results
Active HMGR in tumor cells is required for activation of TCR γγγγδδδδ cells Various cell lines of human origin were tested for the capacity to activate TCR Vγ9-Vδ2 cells in the presence or absence of exogenous IPP and after treatment with ZOL (Table 3).
APC Cell type Medium IPP ZOL
Daudi Bone marrow, B-cell lymphoma 2527* 14342 5647
THP-1 Bone marrow, monocytes 197 3135 16393
CEM 1.3 Bone marrow, T-cell lymphoma 310 6586 1564
K562 Bone marrow, erythroleukemia 36 2762 1145
A-375 Skin, melanoma 188 9169 15842
A-431 Skin, epidermoid carcinoma 89 3269 3067
Colo 201 Colon epithelia, coloncarcinoma 260 3103 4374 HEP G2 Liver parenchyma, hepatocarcinoma 96 12040 12417
HuH6 Liver parenchyma, hepatoblastoma 80 4329 3451
A-243 Central nervous system, astrocytoma 23 5415 7141 U118 Central nervous system, glioblastoma 33 2114 5575 BS 125.3.2 Central nervous system, glioblastoma 46 13942 8440 MRK-nu-1 Mammary gland, mammary carcinoma 35 3003 1965 HMC-1-8 Pleural effusion, mammary carcinoma 17 2011 1845 YMB-1 Mammary gland, mammary carcinoma 1162 3695 1525 Fibroblasts isolated from primary, lung connective tissue 175 5085 8768
Table 3. Tumor cell lines of different tissue origin and primary lung fibroblasts were used as APC. Cells were either incubated with medium or IPP (10 µM) or were pulsed with ZOL (zoledronate, 50 µg/ml) for 3 h before T cell were added. * Numbers represent mean values in pg/ml of TNFα release by the G2B9 TCR γδ clone.
We found that not only Daudi cell but also YMB-1 cells, a solid breast carcinoma activate TCR γδ cells (Table 3). In order to investigate whether HMGR is involved in generation of TCR γδ stimulatory antigens we treated both, Daudi and YMB-1 cells with mevastatin (MEV), and inhibitor of the HMGR catalytic site (Istvan and Deisenhofer, 2001) 2 h before TCR γδ cells addition. Activation of TCR Vγ9-Vδ2 was strongly reduced in the presence of MEV treated Daudi or YMB-1 cells (Figure 11). To rule out toxicity and unspecific inhibitory effect of MEV stimulation with exogenous IPP and PHA was also performed (Figure 11).
Figure 11. TCR γδ cell activation, by tumor cells, depends on active HMGR.
Daudi or YMB-1 cells, in the absence or presence of MEV, were used as APC in TCR γδ cell activation assay. In control experiment stimulation with exogenously added IPP or PHA was performed.
HMGR is one of the most tightly regulated enzyme in the cells (Goldstein and Brown, 1990). The activity of HMGR is controlled through protein synthesis, degradation and phosphorylation (Cheng et al., 1999). In order to further
investigate the involvement of HMGR in the generation of TCR γδ stimulatory
ligands we treated Daudi cells with 7-DHC or farnesol, two endogenous metabolites that facilitate degradation of HMGR (Correll et al., 1994; Honda et al., 1998). Both compounds induced reduction of intracellular HMGR protein levels (Figure 12A) and significantly inhibited TCR γδ activation by Daudi cells (Figure 12B). In control experiments TCR γδ cell activation was not inhibited when stimulated with exogenous IPP in the presence of these compounds (Figure 12B).
Figure 12. TCR γδ cell activation by tumor cells depends on the HMGR protein level.
(A) HMGR protein levels in lysates after treatment of Daudi cells with 7-DHC or farnesol detected by immunoprecipitation and Western blot.
(B) Daudi cells treated either with 7-DHC or farnesol were used to stimulate TCR γδ cells. As control, activation in the presence of exogenously added IPP was performed.
These experiments show that active HMGR is important for stimulation of TCR γδ cells by Daudi and YMB-1 cells. Moreover, the data suggest that indeed
phosphorylated metabolites generated in the mevalonate pathway are the stimulatory ligands for TCR Vγ9-Vδ2 cells.
HMGR overexpressing cells are potent TCR γγγγδδδδ cells stimulators Transfected cells expressed high levels of HMGR as shown by intracellular staining with a specific HMGR mAb (Figure 13)
Figure 13. HMGR overexpression in transfected Daudi cells.
Intracellular level of HMGR protein in HMGR-trasfected (bold line) or non-transfected (thin line) Daudi cells was determined by staining with anti-HMGR specific mAb. Control staining was performed with irrelevant mAb (dotted lines).
These cells were used as APC in TCR γδ activation assay. The capacity to stimulate TCR γδ cells by Daudi-HMGR transfected cells was significantly
increased as compared to the wild type Daudi cells (Figure 14). Moreover, when APC were treated with suboptimal doses of MEV, T cell activation was strongly
reduced. The complete inhibition, in case of Daudi-HMGR cells, was achieved only when MEV was added at least 12 h before the assay (Figure 14). HMGR- transfected cells might require longer incubation with inhibitor since the HMGR protein level in these cells is increased and also mevalonate metabolites might reach higher concentrations.
Figure 14. HMGR overexpression increases TCR γδ cell activation.
HMGR-transfected (closed bars) or wt Daudi cells (open bars) were used as APC in TCR γδ stimulation assay. A suboptimal dose (10 µM) of MEV was added 2, 6 or 12 h before incubation with T cells. Stimulation with exogenouse IPP was used as positive control.
Nitrogen-containing bisphosphonates treated APC activate TCR γγγγδδδδ cells The involvement of endogenous mevalonate pathway in the generation of TCR γδ stimulatory ligands was further investigated in a series of experiments with nitrogen-containing bisphosphonate drugs (nBP). These drugs have a
capacity to inhibit farneslypyrophosphate-synthase (FPP-synthase), (Thompson et al., 2002), (one of the enzymes in the mevalonate pathway, Figure 5B) and in vitro lead to accumulation of IPP/DMAPP (Bergstrom et al., 2000) metabolites that are able to activate TCR γδ cells when exogenously added (Selin et al., 1992; Burk et al., 1995; Tanaka et al., 1995).
We tested two structurally different nBP, zoledronate (ZOL) and pamidronate (PAM) in TCR γδ activation assay. We compared their effect with etidronate, a non-nBP compound that does not block FPP-synthase (Bergstrom et al., 2000;
Dunford et al., 2001). APC treated with either ZOL or PAM, but not with etidronate, stimulated TCR γδ cells very efficiently (Figure 15).
Figure 15. nBP treated APC stimulate TCR γδ cells.
Daudi cells treated with increasing doses of ZOL () or PAM () or etidronate () were used as APC in TCR γδ activation assay.
